红肉火龙果液泡转化酶基因HpVIN1的克隆、表达和酶活性鉴定  

Cloning,Expression and Enzyme Activity Identification of the Vacuo-lar Invertase Gene HpVIN1 from Red Pitaya

作　　者：郑乾明 晏霜 王红林 解璞[1] 马玉华[2] ZHENG Qianming;YAN Shuang;WANG Honglin;XIE Pu;MA Yuhua(Guizhou Institute of Pomology Science,Guizhou Academy of Agricultural Sciences,Guiyang,Guizhou 550006,China;Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Region,Ministry of Agriculture and Rural Affairs,Guizhou Academy of Agricultural Sciences,Guiyang,Guizhou 550006,China)

机构地区：[1]贵州省农业科学院贵州省果树科学研究所,贵州贵阳550006 [2]贵州省农业科学院农业农村部喀斯特山区作物基因资源与种质创新重点实验室,贵州贵阳550006

出　　处：《热带作物学报》2024年第12期2524-2533,共10页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.32060674);贵州省农业科学院国家自然科学基金后补助项目(黔农科院国基后补助[2021]61);贵州省科技计划项目(黔科合中引地[2023]033)。

摘　　要：液泡转化酶(vacuolar invertase,VIN)不可逆地分解蔗糖生成葡萄糖和果糖,是可溶性糖代谢的关键酶,参与植物的生长发育、产量品质形成和抵御逆境。为探讨VIN基因在红肉火龙果(Hylocereus polyrhizus)可溶性糖代谢中的生理功能,从果实中克隆HpVIN1基因,开展序列比对、系统进化、基因表达、亚细胞定位、酵母生长互补分析和蔗糖分解活性检测。基于RT-PCR(reverse transcription-polymerase chain reaction)扩增获得HpVIN1基因,其开放阅读框(open reading frame,ORF)长度为1935 bp,编码644个氨基酸。序列分析表明,HpVIN1含有转化酶催化蔗糖分解的关键结构域,且在N端含有液泡定位的相关序列。系统进化分析表明HpVIN1与猕猴桃、枇杷、苹果和葡萄的VINs具有较近的亲缘关系。实时荧光定量PCR检测表明,HpVIN1在茎和果实转色期(花后20~25 d)的表达量较高,果实成熟期(花后30 d)表达微弱。HpVIN1融合绿色荧光蛋白在拟南芥叶肉原生质体瞬时表达表明HpVIN1定位于液泡膜和液泡。HpVIN1在蔗糖利用缺陷的酵母株系过表达,恢复酵母以蔗糖为唯一碳源的生长,证明HpVIN1具有蔗糖分解活性。酵母总蛋白的离体催化实验表明,HpVIN1分解蔗糖产生葡萄糖和果糖,蔗糖分解活性在pH 4.0时最大,并随pH的升高快速降低。研究结果表明,位于液泡内的HpVIN1具有分解蔗糖产生葡萄糖和果糖的酶活性,参与红肉火龙果茎和果实转色期的可溶性糖代谢。Vacuolar invertase(VIN),degrading sucrose to produce glucose and fructose irreversibly,is a key enzyme in soluble sugar metabolism,and involved in plant growth and development,yield and quality formation,and stress resistance.To investigate the physiological functions of VIN genes in soluble sugar metabolism of red pitaya(Hylocereus polyrhizus),HpVIN1 gene was cloned from the fruit,and sequence comparison,phylogenetic relationship,gene expression,subcellular localization,yeast growth complementation and sucrose degradation activity were analyzed.Based on RT-PCR(reverse transcription-polymerase chain reaction)amplification,the ORF(open reading frame)of HpVIN1 gene with a length of 1935 bp was isolated,which encoded 644 amino acids.Sequence analysis showed that HpVIN1 contained key domains related to sucrose degradation activity of invertase,as well as vacuole localization sequences in the N terminal.Phylogenetic analysis suggested that HpVIN1 was closely related to VIN genes from kiwifruit,loquat,apple and grape.Real time fluorescence quantitative PCR detection revealed that HpVIN1 was highly expressed in stems and fruits during the veraison stage(20~25 days after flowering),and weakly expressed in ripen fruits(30 days after flow-ering).The transient expression of HpVIN1 fused by green fluorescent protein in Arabidopsis thaliana mesophyll pro-toplasts demonstrated that HpVIN1 protein was localized in the tonoplast and vacuole.By over-expressing in the yeast strain with sucrose utilization defects,HpVIN1 could restore the yeast growth using sucrose as the sole carbon source,which proved that HpVIN1 had the sucrose degradation activity.In vitro catalytic experiment using the yeast total pro-tein suggested that HpVIN1 could degrade sucrose into glucose and fructose.The sucrose degradation activity was the highest at pH 4.0,and rapidly decreased as pH value increasing.The results show that HpVIN1 that locates in the vacu-ole,has the enzyme activity of degrading sucrose to produce glucose and fructose,and participates

关 键 词：火龙果 液泡转化酶基因 蔗糖分解 亚细胞定位 酵母表达 

分 类 号：S667.9[农业科学—果树学]